Pairing probiotics and prebiotics, methods for growth and use, separately and in combination

ABSTRACT

A method for growing probiotic organisms wherein the growth media includes prebiotics especially selected and prepared to be paired with the probiotic organisms being grown. The prebiotic formula is optimized to grow the desired probiotic organisms, as well as important byproducts of the growth process. Specialized freeze-drying buffers may also be paired with certain probiotic organisms for the freeze-drying process.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/655,935, filed on Apr. 11, 2018, which is herebyincorporated by reference in its entirety.

BACKGROUND 1. The Field of the Invention

This invention relates to methods for producing probiotics, and moreparticularly to the pairing of particular probiotics and prebiotics tooptimize growth and usefulness of the probiotics.

2. Background

Numerous products contain probiotics for a variety of purposes. Thesepurposes may include regulating digestion, improving and maintaininghealth in general, or improving specific physiological systems, as wellas other purposes. The probiotic Lactobacillus acidophilus is an exampleof the use of probiotics as a nutritional supplement, or as a componentin foods such as yogurts.

Generally, probiotics may be grown or cultured using a broth and/or anagar spread plate. There are various formulations for such a broth andvarious media used for such agar spread plates. These formulations arerelatively standardized for both broths and agar spread plates.

Methods for growing and culturing common probiotics, like Lactobacillusacidophilus, are generally established. For example, an original strainof the desired probiotic may be obtained and used an inoculationmaterial. The inoculation material may be grown in a fermenter (i.e., aliquid broth) and/or on a surface (i.e., an agar plate). This growthgenerally happens under pre-defined and monitored conditions. The grownprobiotic may then be harvested. After being harvested, the probioticmay be blended with other probiotics. The single probiotic or blend ofprobiotics may be preserved in some manner for transportation and/orstorage.

While there are various, useful probiotics that are found in nature, notall probiotics are available for commercial use. What is needed is animproved method or methods for culturing and harvesting more types ofprobiotics. Moreover, to get the full benefit of certain probiotics,those probiotics should be cultured or grown with the most appropriateprebiotics, or prebiotics specifically matched to optimize the growth ofthe desired probiotics. What is needed is an improved method or methodsfor producing or culturing various, useful probiotics to fully realizethe potential and benefits of those probiotics.

BRIEF SUMMARY OF THE INVENTION

In accordance with the foregoing, certain embodiments of a probioticproduct and/or method for production in accordance with the inventionmay provide probiotics that can be used for a variety of purposes,including without limitation, nutritional supplements, topicalapplications, and related uses and products.

There are a number of probiotic organisms that may be considered usefuland helpful to promote or maintain health in humans and animals. Suchprobiotics may include Bacteroides thetaiotaomicron, Bacteroidesfragilis, Bacteroides ovatus, Bacteroides uniformis, Faecalibacteriumprausnitzii, Akkermansia muciniphila, Eubacterium rectale, Collinsellaaerofaciens, Desulfovibrio piger, Clostridium symbiosum, Mycobacteriumvaccae, Eubacterium limosum, Butyricicoccus pullicaecorum, Roseomonasmucosa, Lactobacillus farciminis, Staphylococcus epidermis,Staphylococcus xylosus, Pediococcus acidilactici, Roseburia hominis, andAnaerostipes caccae. These probiotics, and methods for growing them, areprimarily described herein, but the methods and formulations describedherein may be applicable to other probiotic organisms.

A method for growing probiotic organisms, or growing unique probioticfermentations, may comprise a number steps. The method may includeselecting a probiotic organism to grow. The method may include selectinga growth broth for growing the probiotic organism, wherein the growthbroth includes a prebiotic formulation that is optimized for theselected probiotic organism. The method may include inoculating thegrowth broth with the probiotic organism. The method may include growingthe probiotic organism in the growth broth and harvesting the probioticorganism from the growth broth. The method may include selecting an agarspread plate for growing the probiotic organism, wherein the agar spreadplate media includes a prebiotic formula that is optimized for growingthe probiotic organism. The method may include inoculating the agarspread plate with the probiotic organism harvested from the growthbroth. The method may include growing the probiotic organism on the agarspread plate and harvesting the probiotic organism from the agar spreadplate.

A method may further include freeze-drying the harvested probioticorganism with a lyophilization reagent. The lyophilization reagent maybe comprised of one or more of the following: glucosamine, glutamine,sucrose, mannitol, trehalose, glycerol, inositol, raffinose, inulin,powdered skim milk, activated charcoal, soluble starch, collagen powder,chondroitin sulfate, glucosamine sulfate, guar, acacia, silica, andfructooligosaccharide P95.

A method as described herein may be used for growing any of theprobiotic organisms included, as well as other probiotic organisms. Amethod may include the use of a specific formulation of prebiotics thatare optimized for, or preferentially paired with, specific probioticorganisms. This pairing of certain prebiotics with certain probioticshelps promote better and faster growth of the desired probiotic. The useof certain, important prebiotics can promote the growth of a probioticorganism and specific byproducts of importance. Thus, a prebiotic, orprebiotic formulation, may be selected to be paired with a probioticorganism both for growing the probiotic organism and a desiredbyproduct, or group of byproducts, that may be helpful or important fora given purpose.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing features of the present invention will become more fullyapparent from the following description and appended claims, taken inconjunction with the accompanying drawings and experimental data.Understanding that these drawings and date depict only typicalembodiments of the invention and are, therefore, not to be consideredlimiting of its scope, the invention will be described with additionalspecificity and detail through use of the accompanying drawings and datain which:

FIG. 1 illustrates a table providing results of measuring the amount ofcertain short chain fatty acids produced from certain probioticorganisms, where the values for short chain fatty acids are inmilligrams per milliliter measured from culture supernatant; and

FIG. 2 is a schematic diagram of an embodiment of a method for growingprobiotic organisms.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It will be readily understood that the components of the presentinvention, as generally described herein, could be arranged and designedin a wide variety of different configurations or formulations. Thus, thefollowing more detailed description of the embodiments of the system,product and method of the present invention, is not intended to limitthe scope of the invention, as claimed, but is merely representative ofvarious embodiments of the invention.

In one embodiment, a method for growing or culturing numerous probioticorganisms may include the following steps: selecting a probioticorganism, or a group of probiotic organisms, to grow; selecting a growthbroth, which may include a prebiotic formulation specially designed tohelp grow the probiotic organism selected; growing the probioticorganism in the growth broth; harvesting the probiotic organism from thegrowth broth; selecting a growth plate, which may include a prebioticformulation specially designed to help grow the probiotic organismselected; growing the probiotic organism on the growth plate; andharvesting the probiotic organism from the growth plate.

A probiotic organism, or a group of organisms, grown in this manner canbe freeze-dried after harvesting to prepare the organism for transferand/or storage.

The various steps in the process for growing a probiotic organism mayinclude variations at every level. For example, and not by way oflimitation, a growth broth may or may not include a prebiotic formulaspecially selected for the probiotic organism being grown. A prebioticformula may be specifically designed for one or more probioticorganisms, thereby allowing for numerous and varied formulations forprebiotic formulas. Also, the conditions for growing certain probioticorganisms can vary.

Generally, a probiotic organism grown in accordance with the disclosedmethod will be one or more of the following: Bacteroidesthetaiotaomicron, Bacteroides fragilis, Bacteroides ovatus, Bacteroidesuniformis, Faecalibacterium prausnitzii, Akkermansia muciniphila,Eubacterium rectale, Collinsella aerofaciens, Desulfovibrio piger,Clostridium symbiosum, Mycobacterium vaccae, Eubacterium limosum,Butyricicoccus pullicaecorum, Roseomonas mucosa, Lactobacillusfarciminis, Staphylococcus epidermis, Staphylococcus xylosus,Pediococcus acidilactici, Roseburia hominis, and Anaerostipes caccae.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 26 grams of tryptic soy broth, approximately4 grams of inulin, and approximately 0.4 grams of cysteine inapproximately 900 milliliters of water. Such a growth broth may be keptand/or used in a sealed one liter bottle. Such a growth broth may beused to grow a probiotic organism, for example and not by way oflimitation, Bacteroides fragilis, Bacteroides ovatus, Bacteroidesuniformis, Eubacterium limosum, and Clostridium symbiosum.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 26 grams of tryptic soy broth, approximately4 grams of inulin, approximately 0.4 grams of cysteine, andapproximately 3.6 milliliters of 50% glycerol in approximately 900milliliters of water. Such a growth broth may be kept and/or used in asealed one liter bottle. Such a growth broth may be used to grow aprobiotic organism, for example and not by way of limitation,Bacteroides thetaiotaomicron.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 26 grams of tryptic soy broth, approximately4 grams of inulin, approximately 2.5 grams of Middlebrook 7H9 broth, andapproximately 7.2 milliliters of 50% glycerol in approximately 900milliliters of water. Such a growth broth may be kept and/or used in asealed one liter bottle. Such a growth broth may be used to grow aprobiotic organism, for example and not by way of limitation,Mycobacterium vaccae. This probiotic organism may be grown aerobically.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 50 grams of MRS broth, approximately 0.1grams of glutathione, approximately 0.4 grams of cysteine, andapproximately 0.2 grams of uric acid in approximately 900 milliliters ofwater. Such a growth broth may be kept and/or used in a sealed one literbottle. Such a growth broth may be used to grow a probiotic organism,for example and not by way of limitation, Collinsella aerofaciens.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 50 grams of MRS broth in approximately 900milliliters of water. Such a growth broth may be kept and/or used in asealed one liter bottle. Such a growth broth may be used to grow aprobiotic organism, for example and not by way of limitation,Pediococcus acidilactici and Lactobacillus farciminis. These probioticorganisms may be grown aerobically or anaerobically.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 33 grams of brain heart infusion,approximately 0.1 grams of glutathione, approximately 0.4 grams ofcysteine, approximately 8 grams of inulin, approximately 4.5 grams ofyeast extract, and approximately 1.8 grams of sodium acetate inapproximately 900 milliliters of water. Such a growth broth may be keptand/or used in a sealed one liter bottle. Such a growth broth may beused to grow a probiotic organism, for example and not by way oflimitation, Faecalibacterium prausnitzii.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 7 grams of nutrient broth, approximately 4.5grams of tryptone, approximately 9 grams of yeast extract, approximately8 grams of inulin, approximately 4.5 grams of dextrose, approximately1.5 grams of dipotassium phosphate (K₂HPO₄), approximately 4.5milliliters of 20% tween 80, approximately 0.4 grams of cysteine, andapproximately 27 milliliters of a salt solution in approximately 900milliliters of water. The salt solution may be comprised ofapproximately 0.2 grams of calcium chloride (CaCl₂), approximately 0.24grams of magnesium sulfate (MgSO₄), approximately 1.0 grams ofmonopotassium phosphate (KH₂PO₄), approximately 10 grams of sodiumbicarbonate (NaHCO₃), and approximately 2.0 grams of sodium chloride(NaCl) in approximately one liter of water. Such a growth broth may bekept and/or used in a sealed one liter bottle. Such a growth broth maybe used to grow a probiotic organism, for example and not by way oflimitation, Eubacterium rectale, Anaerostipes caccae, and Butyricicoccuspullicaecorum.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 33 grams of brain heart infusion,approximately 1.0 grams of mucin, and approximately 0.4 grams ofcysteine in approximately 900 milliliters of water. Such a growth brothmay be kept and/or used in a sealed one liter bottle. Such a growthbroth may be used to grow a probiotic organism, for example and not byway of limitation, Akkermansia muciniphila.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 27 grams of tryptic soy broth, approximately4 grams of yeast extract, approximately 0.8 grams of sodium lactate,approximately 0.4 grams of magnesium sulfate, approximately 0.3 grams offerric ammonium sulfate-6H₂O, approximately 0.4 grams of cysteine, andapproximately 0.1 grams of glutathione in approximately 900 millilitersof water. Such a growth broth may be kept and/or used in a sealed oneliter bottle. Such a growth broth may be used to grow a probioticorganism, for example and not by way of limitation, Desulfovibrio piger.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 50 grams of MRS broth, approximately 0.4grams of cysteine, approximately 0.1 grams of glutathione, andapproximately 27 milliliters of a salt solution in approximately 900milliliters of water. The salt solution may be comprised ofapproximately 0.2 grams of calcium chloride (CaCl₂), approximately 0.24grams of magnesium sulfate (MgSO₄), approximately 1.0 grams ofmonopotassium phosphate (KH₂PO₄), approximately 10 grams of sodiumbicarbonate (NaHCO₃), and approximately 2.0 grams of sodium chloride(NaCl) in approximately one liter of water. Such a growth broth may bekept and/or used in a sealed one liter bottle. Such a growth broth maybe used to grow a probiotic organism, for example and not by way oflimitation, Roseburia hominis.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 7 grams of nutrient broth in approximately900 milliliters of water. Such a growth broth may be kept and/or used ina sealed one liter bottle. Such a growth broth may be used to grow aprobiotic organism, for example and not by way of limitation, Roseomonasmucosa.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 27 grams of tryptic soy broth inapproximately 900 milliliters of water. Such a growth broth may be keptand/or used in a sealed one liter bottle. Such a growth broth may beused to grow a probiotic organism, for example and not by way oflimitation, Staphylococcus epidermidis. This probiotic organism may begrown aerobically or anaerobically.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 27 grams of tryptic soy broth andapproximately 7.2 milliliters of 50% glycerol in approximately 900milliliters of water. Such a growth broth may be kept and/or used in asealed one liter bottle. Such a growth broth may be used to grow aprobiotic organism, for example and not by way of limitation,Staphylococcus xylosus. This probiotic organism may be grown aerobicallyor anaerobically.

In one embodiment, an agar spread plate media, or agar spread plate, maybe comprised of approximately 40 g/L tryptic soy agar, approximately 50ml/L defibrinated sheep blood, approximately 0.5 g/L cysteine, andapproximately 0.1 g/L glutathione. Such an agar spread plate may be usedto grow a probiotic organism, for example and not by way of limitation,Bacteroides thetaiotaomicron, Bacteroides fragilis, Bacteroides ovatus,Bacteroides uniformis, Eubacterium limosum, Clostridium symbiosum,Collinsella aerofaciens, Faecalibacterium prausnitzii, Eubacteriumrectale, Roseburia hominis, Anaerostipes caccae, Butyricicoccuspullicaecorum, Roseomonas mucosa, Staphylococcus epidermidis, andStaphylococcus xylosus.

In one embodiment, an agar spread plate media, or agar spread plate, maybe comprised of approximately 12 g/L agar and a growth broth(approximately 3 g/L) comprised of approximately 26 grams of tryptic soybroth, approximately 4 grams of inulin, approximately 2.5 grams ofMiddlebrook 7H9 broth, and approximately 7.2 milliliters of 50% glycerolin approximately 900 milliliters of water. Such an agar spread plate maybe used to grow a probiotic organism, for example and not by way oflimitation, Mycobacterium vaccae. This probiotic organism may be grownaerobically.

In one embodiment, an agar spread plate media, or agar spread plate, maybe comprised of approximately 70 g/L of MRS agar. Such an agar spreadplate may be used to grow a probiotic organism, for example and not byway of limitation, Pediococcus acidilactici and Lactobacillusfarciminis. These probiotic organisms may be grown aerobically oranaerobically.

In one embodiment, an agar spread plate media, or agar spread plate, maybe comprised of approximately 12 g/L agar and a growth broth(approximately 3 g/L) comprised of approximately 33 grams of brain heartinfusion, approximately 1.0 grams of mucin, and approximately 0.4 gramsof cysteine in approximately 900 milliliters of water. A pour plate mayuse 8 g/L agar. Such an agar spread plate may be used to grow aprobiotic organism, for example and not by way of limitation,Akkermansia muciniphila.

In one embodiment, an agar spread plate media, or agar spread plate, maybe comprised of approximately 10 g/L agar and a growth broth(approximately 3 g/L) comprised of approximately 27 grams of tryptic soybroth, approximately 4 grams of yeast extract, approximately 0.8 gramsof sodium lactate, approximately 0.4 grams of magnesium sulfate,approximately 0.3 grams of ferric ammonium sulfate-6H₂O, approximately0.4 grams of cysteine, and approximately 0.1 grams of glutathione inapproximately 900 milliliters of water. A pour plate may use 10 g/Lagar. Such an agar spread plate may be used to grow a probioticorganism, for example and not by way of limitation, Desulfovibrio piger.

In one embodiment, a freeze-drying buffer, or lyophilization buffer, maybe used to freeze-dry probiotic organisms that are grown and harvested.Freeze-drying reagents, or lyophilization reagents, may include thefollowing compounds alone or in combination: glucosamine, sucrose,mannitol, trehalose, glycerol, inositol, raffinose, inulin, powderedskim milk, activated charcoal, soluble starch, collagen powder,chondroitin sulfate, glucosamine sulfate, glutamine, guar, acacia,silica, and fructooligiosaccharide P95. A freeze-drying buffer, orlyophilization buffer, may be comprised of appropriate amounts of one ormore of the freeze-drying reagents in deionized water, which is thenadjusted to a pH of 7.0 with potassium hydroxide.

In one embodiment, a freeze-drying buffer may be made at least one dayprior to the freeze-drying process. A freeze-drying buffer may be storedovernight in an anaerobic chamber to allow dissolved oxygen to escapethe solution. The volume of a freeze-drying buffer used may be equal tothe volume of a cell pellet or probiotic organisms obtained aftercentrifugation.

In one embodiment, a freeze-drying buffer or media may be made withanti-oxidant and growth factors, for example and not by way oflimitation, cysteine, ascorbic acid, glutathione, uric acid, riboflavin,glutamic acid, sodium sulfite, and quinones.

In one embodiment, a freeze-drying buffer, or lyophilization buffer, maybe comprised of 10% mannitol, 12.5% sucrose, 2.5% trehalose, 1.0%glycerol, 10% skim milk, 0.02% cysteine, 0.01% ascorbic acid, 0.005%glutathione, 0.005% uric acid, and 2.0% charcoal in deionized water andadjusted to pH 7 with potassium hydroxide. The percentages provided maybe considered approximate percentages. This embodiment of afreeze-drying buffer will hereinafter be referred to as “Freeze-dryingBuffer A.” Freeze-drying Buffer A may be used with various probioticorganisms, for example and not by way of limitation, Mycobacteriumvaccae, Pediococcus acidilactici, Roseomonas mucosa, Lactobacillusfarciminis, Staphylococcus epidermidis, and Staphylococcus xylosus.

In one embodiment, a freeze-drying buffer, or lyophilization buffer, maybe comprised of 10% mannitol, 12.5% sucrose, 2.5% trehalose, 1.0%glycerol, 10% skim milk, 0.2% cysteine, 0.1% ascorbic acid, 0.05%glutathione, 0.05% uric acid, and 2.0% charcoal in deionized water andadjusted to pH 7 with potassium hydroxide. The percentages provided maybe considered approximate percentages. This embodiment of afreeze-drying buffer will hereinafter be referred to as “Freeze-dryingBuffer B.” Freeze-drying Buffer B may be used with various probioticorganisms, for example and not by way of limitation, Bacteroidesthetaiotaomicron, Bacteroides fragilis, Bacteroides ovatus, Bacteroidesuniformis, Eubacterium limosum, Collinsella aerofaciens, Akkermansiamuciniphila, and Roseburia hominis.

In one embodiment, a freeze-drying buffer, or lyophilization buffer, maybe comprised of 10% mannitol, 10% inulin, 5% short chain FOS, 10%soluble starch, 2.5% trehalose, 1.0% glycerol, 2.0% cysteine, 0.5%ascorbic acid, 0.5% glutathione, 0.1% uric acid, and 6.0% charcoal indeionized water and adjusted to pH 7 with potassium hydroxide. Thepercentages provided may be considered approximate percentages. Thisembodiment of a freeze-drying buffer will hereinafter be referred to as“Freeze-drying Buffer C.” Freeze-drying Buffer C may be used withvarious probiotic organisms, for example and not by way of limitation,Clostridium symbiosum, Faecalibacterium praunsnitzii, Eubacteriumrectale, Anaerostipes caccae, and Butyricicoccus pullicaecorum.

In one embodiment, a freeze-drying buffer, or lyophilization buffer, maybe comprised of 10% mannitol, 12.5% sucrose, 2.5% trehalose, 10%collagen powder, 1.0% chondroitin sulfate, 1.0% glycerol, 2.0% cysteine,0.5% ascorbic acid, 0.5% glutathione, 0.1% uric acid, and 6.0% charcoalin deionized water and adjusted to pH 7 with potassium hydroxide. Thepercentages provided may be considered approximate percentages. Thisembodiment of a freeze-drying buffer will hereinafter be referred to as“Freeze-drying Buffer D.” Freeze-drying Buffer D may be used withvarious probiotic organisms, for example and not by way of limitation,Desulfovibrio piger.

In one embodiment, a dilution buffer, or anti-oxidant resuspensionsolution, may be utilized for plating lyophilized bacteria and/orobtaining plate counts and estimating cfu/gram (colony-forming unit pergram of sample). In one embodiment, a dilution buffer, or anti-oxidantresuspension solution, may be comprised of 0.2% cysteine, 0.1% ascorbicacid, 0.05% glutathione, 0.05% uric acid, and 0.0425% monopotassiumphosphate (KH₂PO₄) monopotassium phosphate (KH₂PO₄) in deionized waterand adjusted to pH 7 with potassium hydroxide solution.

Culture conditions for various probiotic organisms have been determinedexperimentally.

In one embodiment, various probiotic organisms, for example and not byway of limitation, Bacteroides thetaiotaomicron, Bacteroides fragilis,Bacteroides ovatus, Bacteroides uniformis, Eubacterium limosum,Collinsella aerofaciens, Akkermansia muciniphila, and Desulfovibriopiger require 48-96 hours of incubation at 37° C. to achieve the desiredgrowth. The addition of anti-oxidants during media preparation may berequired, but no special anaerobic conditions are required for growth inliquid media, or growth broth. The liquid media bottles should be cappedto prevent contamination and to limit airflow to the growing probioticcultures. Disturbance of the liquid media, such as swirling, should beavoided to limit oxygen rich air being mixed with the liquid media.

Centrifugation and freeze-drying processes can be performed in anaerobic environment if done quickly, for example, by mixing thecentrifuged pellet with a freeze-dry buffer immediately after pouringoff the supernatant and then placing the suspension at −20° C. It maytake approximately one hour to fully freeze the suspension. Then thesuspension can be placed in the lyophilizer.

Spread plating or pour plating techniques can be used for theseprobiotic organisms, provided that the dilution buffer, or antioxidantresuspension solution, is used. Plates should be put in an anaerobicchamber or airtight box containing anaerobic gas producing packets.Growth of colonies on plates typically requires 24-72 hours at 37° C.

In one embodiment, various probiotic organisms, for example and not byway of limitation, Clostridium symbiosum, Faecalibacterium prausnitzii,Eubacterium rectale, Roseburia hominis, Anaerostipes caccae, andButyricicoccus pullicaecorum require 24-48 hours of incubation at 37° C.to achieve desired growth. These probiotic organisms grow poorly unlessincubated in an oxygen-free environment. Oxygen rich air must beevacuated from liquid media bottles before placing them in an anaerobicchamber.

For centrifugation, liquid cultures must be poured into airtightcentrifuge bottles and sealed in an anaerobic chamber. The centrifugebottles can then be removed from the chamber to undergo centrifugation.They must be placed back in the anaerobic chamber before opening to pouroff the supernatant. The cell pellets are then suspended in freeze-drybuffer that has been sitting in the anaerobic chamber overnight, or forapproximately twelve hours, to help pull off oxygen dissolved in thebuffer. The cell suspension is then sealed in an airtight container andplaced at −20° C. until lyophilized in aerobic conditions.

All plating techniques must be performed in the anaerobic chamber.Growth of colonies on plates typically requires 24-48 hours at 37° C.

In one embodiment, various probiotic organisms, for example and not byway of limitation, Mycobacterium vaccae, Pediococcus acidilactici,Roseomonas mucosa, Lactobacillus farciminis, Staphylococcus epidermidis,and Staphylococcus xylosus require 24-48 hours of incubation at 37° C.to achieve desired growth, except that M. vaccae may require 5-8 days.These probiotic organisms may be cultured aerobically. Aeration ofliquid media, or growth broth, using a stir plate and a stir barimproves growth of M. vaccae, R. mucosa, and S. xylosus.

Centrifugation, any freeze-drying process, and plate growth techniquesmay all occur in an aerobic environment. Colonies on plates aretypically observed in 24-48 hours at 37° C., except that M. vaccae maytake 3-5 days.

Prebiotic formulations may be used to supplement, support, and optimizeprobiotic growth, which process may be referred to as synbiotics. Inorder to facilitate growth of a given probiotic organism, the correct ormatching prebiotic is crucial. Since probiotic organisms vary in genusand species, specific prebiotic preparations or formulations may berequired. Some prebiotics colonize the lumen and others the mucuslayers. Many prebiotics are butyrate-producing Firmicutes members.

Prebiotics often enhance mucosal butyrate, which has been shown topossibly stimulate the release of mucosal butyrate producers towards thelumen. A process connected with that may affect the intestinalinflammation and consumption of prebiotic compounds.

Some probiotic organisms require other special conditions likeco-culturing with another commensal organism. For example and not by wayof limitation, Bacteroides fragilis produces GABA (Gamma-AminobutyricAcid), a metabolite required by a second organism. Therefore, the twoorganisms may be considered co-dependent.

Various techniques may be utilized to introduce a prebiotic, or aprebiotic formulation, to a method for growing a certain probioticorganism, or multiple probiotic organisms. In one embodiment, a growthmedia may be exogenously supplemented with the needed metabolite. Inanother embodiment, the supernatant of one organism may be used tosupplement the growth media used for growing another organism ofinterest. For example and not by way of limitation, growth ofFaecalibacterium prausnitzii may require special supplementation of oneor more specific vitamin K metabolites.

Table 1 below describes a prebiotic formulation that may be used withprobiotic organisms including Faecalibacterium prausnitzii,Mycobacterium vaccae, and Lactobacillus farciminis.

TABLE 1 Prebiotic mg/ser Galacto-oligosaccharide 3000.0Isomalt-oligosaccharide 3000.0 IgY-immunoglobulin 100.0 Deoxynojirimycinpolysaccharide (DPM) 500.0 Lactoferrin 50.0 Guggulipids 500.0 Sorghum2000.0 Pectin 1000.0 Fructooligosaccharide (FOS) 1000.0 Blueberry powder1000.0 Black currant 1000.0 Lions mane mushroom 500.0 Cordycepsmilitarus 500.0 Karaya gum 500.0 Pomegranate (ellagitanin) 500.0Geranium thunbergii (dehydroellagitanin) 500.0 Phyllanthus muellerianus(Geraniin and furosin) 500.0 Raspberry extract (gallic ellagic) 500.0Total 16650.0

The amounts in Table 1 may be considered approximations. The prebioticformulation represented in Table 1 may be hereinafter referred to as“Prebiotic Formula Alpha.”

Table 2 below describes a prebiotic formulation that may be used withprobiotic organisms including Eubacterium limosum, Bacteroidesthetaiotaomicron, Lactobacillus vaginalis, and Bacteroides uniformis.

TABLE 2 Prebiotic mg/ser Glycomacropeptide 1000.0 Xylo-oligosaccharide1000.0 Glucomannan 1000.0 Inulin; sprouted greens 1000.0 Galactomannan1000.0 Blueberry powder 1000.0 Thymus 1000.0 Mulberry leaves 1000.0Myo-inositol 1000.0 Modified citrus pectin 1000.0 Tapioca 500.0Glucosaminoglycan 1000.0 Guar sun-fiber 500.0 Oat oligosaccharide 500.0Clitoria ternatea (blue pea flower) 500.0 Cranberry extract 500.0Bearberry (Arctostaphylos uva-ursi) 500.0 Total 14000.0

The amounts in Table 2 may be considered approximations. The prebioticformulation represented in Table 2 may be hereinafter referred to as“Prebiotic Formula Beta.”

Table 3 below describes a prebiotic formulation that may be used withprobiotic organisms including Bacteroides ovatus, Anaerostipes caccae,Staphylococcus xylosus, Staphylococcus epidermidis, and Roseomonasmucosa.

TABLE 3 Prebiotic mg/ser 2′ fructosyllactose 2000.0Isomato-oligosaccharide 1000.0 Fructooligosaccharide Yaconroot/Beneo/innulin 1000.0 Galactooligosaccharide 1000.0 Rutin 1000.0 Oatoligosaccharide 1000.0 Sialylated bovine milk oligosaccharides (S-BMO)1000.0 IgY immunoglobulin 1000.0 Beta glucan 1000.0 Maca 1000.0Lactooligosaccharide 1000.0 Medium Chain Triglycerides 500.0Phosphatidyl choline 500.0 Rose hips/organic green banana 500.0 Citrusbioflavonoids 500.0 Colostrum 500.0 Total 14500.0

The amounts in Table 3 may be considered approximations. The prebioticformulation represented in Table 3 may be hereinafter referred to as“Prebiotic Formula Gamma.”

Table 4 below describes a prebiotic formula that may be used withprobiotic organisms including Collinsella aerofaciens, Bacteroidesovatus, Bacteroides uniformis, Clostridium symbiosum, Bacteroidesfragilis, and Roseburia hominis.

TABLE 4 Prebiotic mg/ser Galactooligosaccharide 2000.0Mannan-oligosaccharide 1000.0 Resveratrol 1000.0 Hesperetin 1000.0Arabinoxylan oligosaccharides 1000.0 Dragon fruit 1000.0 Beechwoodextract xylan 1000.0 Arabinogalactan (Larix laricina) 1000.0 DAOantihistamine 1000.0 Xanthan gum 1000.0 Resistant starch 1000.0 Baobabfruit 500.0 Kongorobi berry 1000.0 Resveratrol/Pterostilbene 500.0Curcumin 500.0 Indian Tinospora (Tinospora cordifolia)(stem and root)500.0 Poria mushroom 500.0 Total 15500.0

The amounts in Table 4 may be considered approximations. The prebioticformulation represented in Table 4 may be hereinafter referred to as“Prebiotic Formula Delta.”

The various prebiotic formulations may be utilized with a liquid mediaor growth broth. The prebiotic formulation selected and paired with agiven probiotic organism may constitute approximately 1%-15% of thegrowth broth. Similarly, the various prebiotic formulations may beutilized with an agar spread plate. The prebiotic formulation selectedand paired with a given probiotic organism may constitute approximately1%-15% of the agar spread plate media.

Prebiotic ingredients used in prebiotic formulations can many differenttypes of compounds. For example and not by way of limitation, complexplant oligosaccharides may include the following:Galacto-oligosaccharide; Isomalt-oligosaccharide;Mannan-oligosaccharide; Apple pectin oligosaccharide;Xylo-oligosaccharide; and Fructo-oligosaccharide-inulin. These complexplant oligosaccharides have been shown increase caecal mucin levels byapproximately six-fold and correspond with even higher butyrate levelsand higher abundances of the same mucosal butyrate producers.

Other complex plant oligosaccharides include Oat-oligosaccharides andArabinoxylan-oligosaccharides (AXOS), which have been shown to be apromising class of prebiotics that stimulate the growth ofBifidobacterium longum, an acetate producer to stimulate Eubacteriumrectale, an acetate-converting butyrate producer.

Another complex plant oligosaccharide is Deoxynojirimycin polysaccharide(DPM), which has been shown to increase the Bacteroides to Firmicutesratios, significantly inhibiting the growth of Prevotella, andincreasing the relative abundance of Bacteroides, Lactobacillus,Bifidobacterium, and Akkermansia in mice.

Animal based oligosaccharides may include the following: N-acetylglucosamine-amino glycan Lactulose-saccharide; IgY-immunoglobulin; andSialylated bovine milk oligosaccharides (S-BMO).

Other complex prebiotic compounds may include the following:Cyclodextrin-saccharide; Mulberry-cyanadins; Maca-oligosaccharides;polyphenols; Rutin-flavonoid (E. limosum specifically metabolizedflavonoids); mushroom-polysaccharides; aminoglycans; Shilajit (organiccompounds); Resveratrol; and Pterostilbene.

The criteria for pairing or partnering certain probiotic organisms withspecific prebiotics is driven by the metabolites that are produced bythe probiotic organisms. For example and not by way of limitation, themetabolites produced by probiotic organisms that may be evaluated topair or partner those probiotic organisms with specific prebiotics mayinclude the following: small chain fatty acids, such as butyric acid,acetic acid, propionic acid, hexanoic acid, and the like; organic acids,such as lactic acid, malic acid, citric acid, and the like;bacteriocins, which are natural antimicrobials produced by probioticorganisms. The research and analysis required to properly partnerprobiotic organisms and prebiotic compounds can take years to complete.

FIG. 1 provides an example of the research conducted and evaluated toproperly partner probiotic organisms and prebiotic compounds based onthe production of short chain fatty acids produced in selected strainsof probiotic organisms. The values for short chain fatty acids in FIG. 1are in milligrams per milliliter measured from culture supernatant.

In one embodiment, a phage may be used as a terrain biotic. For exampleand not by way of limitation, terrain biotics may include Streptococcusmutans, Actinomyces naeslundi, Cutibacterium acnes, Porphyromonasgingivalis, Treponema denticola, Fusobactium nucleatum, Aggregatibacteractinomycetemcomitans, and Tannerella forsythia.

Referring to FIG. 2, one embodiment of a method 10 for producingprobiotic organisms may include the steps of selecting a probioticorganism to grow 15, selecting a prebiotic formula 20 to pair with theprobiotic organism selected, selecting a growth broth 25, inoculatingthe growth broth 30, growing the probiotic organism 35, harvesting theprobiotic organism from the growth broth 40, selecting a growth plate45, inoculating the growth plate 50, growing the probiotic organism onthe growth plate 55, harvesting the probiotic organism from the growthplate 60, and freeze-drying the probiotic organism 65.

The step of selecting a probiotic organism to grow 15 can be based onnumerous factors. The uses, properties, and metabolites of a givenprobiotic organism may be considered. Generally, the process ofbeginning to grow a probiotic organism will start with a pure strain ofthe organism, but it is possible to grow multiple probiotic organismstogether. However, more care should be taken to select organisms thatcan be grown using the same growth media, as well as the same prebioticformulations, to optimize growth of all probiotic organisms selected.

The step of selecting a prebiotic formula, or formulation, 20 to growthe selected probiotic organism should be based primarily on theprobiotic organism selected. The intention is to match or partner theprebiotics with the probiotic organism as best as can be done to promoteoptimal growth of the probiotic organism. For example and not by way oflimitation, if Mycobacterium vaccae is the probiotic organism selectedto be grown, Prebiotic Formula Alpha would be an optimal choice forprebiotics to pair with the probiotic organism during the growthprocess.

The step of selecting a growth broth 25, or selecting a liquid media forgrowth 25, may include a number of factors. A growth broth can beselected based on the particular probiotic organism to be grown, andthere are certain growth broths that can be used to grow specificprobiotic organisms. Also, a growth broth may include at least some of aprebiotic formulation to promote the growth process. A growth broth maybe augmented to include a prebiotic formula where the prebiotic formulais approximately 1%-15% of the growth broth media. For example and notby way of limitation, if Bacteroides fragilis is the probiotic organismto be grown, the growth broth used (and previously described) may beaugmented to be approximately 10% Prebiotic Formula Delta.

The step of inoculating the growth broth 30 is generally performed byintroducing the selected probiotic organism into the selected andprepared growth broth by any appropriate means.

The step of growing the probiotic organism in the growth broth 35 isaccomplished by any appropriate means. For example and not by way oflimitation, if Bacteroides fragilis is the probiotic organism to begrown, the growth broth may be kept in a sealed bottle and allowed toincubate for 48-96 hours at 37° C. For example and not by way oflimitation, if Mycobacterium vaccae is the probiotic organism to begrown, the growth should take place anaerobically.

The step of harvesting the probiotic organism from the growth broth 40is generally accomplished by any appropriate means that are consistentwith the requirements for the probiotic organism. For example and not byway of limitation, if Mycobacterium vaccae is the probiotic organism tobe grown, this harvesting process should take place anaerobically.

The step of selecting a growth plate 45, or selecting an agar spreadplate for growing the selected probiotic organism 45, may includeconsideration of multiple factors. The type of media used for the growthplate should be compatible with the probiotic organism to be grown.Also, the media of the growth plate may include a prebiotic formulationto promote growth of the probiotic organism. For example and not by wayof limitation, if Bacteroides thetaiotaomicron is the probiotic organismto be grown, the growth plate used, or agar spread plate used (andpreviously described), may be augmented to include approximately 5%Prebiotic Formula Beta.

The step of inoculating the growth plate 50, or inoculating and/orpreparing the agar spread plate 50, is generally performed byintroducing the probiotic organism harvested from the growth broth 40onto the surface of the selected and prepared growth plate by anyappropriate means. For example and not by way of limitation, ifRoseburia hominis is the probiotic organism to be grown, the inoculatingand/or preparing the growth plate should take place anaerobically.

The step of growing the probiotic organism on the growth plate 55, orgrowing the probiotic organism on the agar spread plate 55, isaccomplished by any appropriate means. For example and not by way oflimitation, if Bacteroides fragilis is the probiotic organism to begrown, the growth plate may be allowed to incubate for 24-72 hours at37° C. For example and not by way of limitation, if Mycobacterium vaccaeis the probiotic organism to be grown, the growth should take placeanaerobically.

The step of harvesting the probiotic organism from the growth plate 60is generally accomplished by any appropriate means that are consistentwith the requirements for the probiotic organism. For example and not byway of limitation, if Mycobacterium vaccae is the probiotic organism tobe grown, this harvesting process should take place anaerobically.

The step of freeze-drying the probiotic organism 65 may includeconsideration of a variety of factors. There may be times when thefreeze-drying step is not required or desired. The probiotic organismmay require preparation for storage and/or delivery. Also, a speciallyprepared buffer for the freeze-dry process, or freeze-drying buffer, maybe utilized for lyophilization of a specific probiotic organism. Forexample and not by way of limitation, if Clostridium symbiosum is theprobiotic organism that has been grown, then Freeze-drying Buffer C maybe used to more appropriately freeze-dry that probiotic organism.

The subject invention may be more easily comprehended by reference tothe specific embodiments recited herein, which are representative of theinvention. However, it must be understood that the specific embodimentsare provided only for the purpose of illustration, and that theinvention may be practiced in a manner separate from what isspecifically illustrated without departing from its scope and spirit.

What is claimed and desired to be secured by United States LettersPatent is:
 1. A method for growing probiotic organisms comprising:selecting at least one probiotic organism to grow; selecting a growthbroth for growing the probiotic organism, wherein the growth brothincludes a prebiotic formula that is optimized for growing the probioticorganism; inoculating the growth broth with the probiotic organism;growing the probiotic organism in the growth broth; harvesting theprobiotic organism from the growth broth; selecting an agar spread platefor growing the probiotic organism, wherein the agar spread plate mediaincludes a prebiotic formula that is optimized for growing the probioticorganism; inoculating the agar spread plate with the probiotic organismharvested from the growth broth; growing the probiotic organism on theagar spread plate; and harvesting the probiotic organism from the agarspread plate.
 2. The method of claim 1, further comprising:freeze-drying the probiotic organism harvested from the agar spreadplate by use of a lyophilization reagent, wherein the lyophilizationreagent includes at least two compounds selected from the groupconsisting of glucosamine, glutamine, sucrose, mannitol, trehalose,glycerol, inositol, raffinose, inulin, powdered skim milk, activatedcharcoal, soluble starch, collagen powder, chondroitin sulfate,glucosamine sulfate, guar, acacia, silica, and fructooligosaccharideP95.
 3. The method of claim 1, wherein the at least one probioticorganism to grow is selected from the group consisting ofFaecalibacterium prausnitzii, Mycobacterium vaccae, and Lactobacillusfarciminis, and the prebiotic formula included in the growth broth andin the agar spread plate media is Prebiotic Formula Alpha.
 4. The methodof claim 1, wherein the at least one probiotic organism to grow isselected from the group consisting of Eubacterium limosum, Bacteroidesthetaiotaomicron, Lactobacillus vaginalis, and Bacteroides uniformis,and the prebiotic formula included in the growth broth and in the agarspread plate media is Prebiotic Formula Beta.
 5. The method of claim 1,wherein the at least one probiotic organism to grow is selected from thegroup consisting of Bacteroides ovatus, Anaerostipes caccae,Staphylococcus xylosus, Staphylococcus epidermidis, and Roseomonasmucosa, and the prebiotic formula included in the growth broth and inthe agar spread plate media is Prebiotic Formula Gamma.
 6. The method ofclaim 1, wherein the at least one probiotic organism to grow is selectedfrom the group consisting of Collinsella aerofaciens, Bacteroidesovatus, Bacteroides uniformis, Clostridium symbiosum, Bacteroidesfragilis, and Roseburia hominis, and the prebiotic formula included inthe growth broth and in the agar spread plate media is Prebiotic FormulaDelta.
 7. The method of claim 1, wherein at least two probioticorganisms are selected from the group consisting of Faecalibacteriumprausnitzii, Mycobacterium vacae, and Lactobacillus farciminis, and theprebiotic formula included in the growth broth and in the agar spreadplate media is Prebiotic Formula Alpha.
 8. The method of claim 1,wherein at least two probiotic organisms are selected from the groupconsisting of Eubacterium limosum, Bacteroides thetaiotaomicron,Lactobacillus vaginalis, and Bacteroides uniformis, and the prebioticformula included in the growth broth and in the agar spread plate mediais Prebiotic Formula Beta.
 9. The method of claim 1, further comprising:freeze-drying the probiotic organisms harvested from the agar spreadplate by use of a Freeze-drying Buffer A when the probiotic organism isat least one probiotic organism selected from the group consisting ofMycobacterium vaccae, Pediococcus acidilactici, Roseomonas mucosa,Lactobacillus farciminis, Staphylococcus epidermidis, and Staphylococcusxylosus.
 10. The method of claim 1, further comprising: freeze-dryingthe probiotic organisms harvested from the agar spread plate by use of aFreeze-drying Buffer B when the probiotic organism is at least oneprobiotic organism selected from the group consisting of Bacteroidesthetaiotaomicron, Bacteroides fragilis, Bacteroides ovatus, Bacteroidesuniformis, Eubacterium limosum, Collinsella aerofaciens, Akkermansiamuciniphila, and Roseburia hominis.
 11. The method of claim 1, furthercomprising: freeze-drying the probiotic organisms harvested from theagar spread plate by use of a Freeze-drying Buffer C when the probioticorganism is at least one probiotic organism selected from the groupconsisting of Clostridium symbiosum, Faecalibacterium praunsnitzii,Eubacterium rectale, Anaerostipes caccae, and Butyricicoccuspullicaecorum.
 12. The method of claim 1, further comprising:freeze-drying the probiotic organism harvested from the agar spreadplate by use of a Freeze-drying Buffer D when the probiotic organism isDesulfovibrio piger.
 13. A method for growing a probiotic organismcomprising: selecting a probiotic organism to grow, wherein theprobiotic organism is selected from the group consisting of Bacteroidesovatus, Anaerostipes caccae, Staphylococcus xylosus, Staphylococcusepidermidis, and Roseomonas mucosa; selecting a growth broth for growingthe probiotic organism, wherein the growth broth is comprised of 1%-15%Prebiotic Formula Gamma; inoculating the growth broth with the probioticorganism; growing the probiotic organism in the growth broth; harvestingthe probiotic organism from the growth broth; selecting an agar spreadplate for growing the probiotic organism, wherein the agar spread platemedia is comprised of 1%-15% Prebiotic Formula Gamma; inoculating theagar spread plate with the probiotic organism harvested from the growthbroth; growing the probiotic organism on the agar spread plate; andharvesting the probiotic organism from the agar spread plate.
 14. Themethod of claim 13 further comprising: freeze-drying the probioticorganism.
 15. The method of claim 13, further comprising: freeze-dryingthe probiotic organism by use of a Freeze-drying Buffer A when theprobiotic organism is Staphylococcus epidermidis or Staphylococcusxylosus.
 16. A method for growing a probiotic organism comprising:selecting a probiotic organism to grow, wherein the probiotic organismis selected from the group consisting of Collinsella aerofaciens,Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis,Clostridium symbiosum, and Roseburia hominis; selecting a growth brothfor growing the probiotic organism, wherein the growth broth iscomprised of 1%-15% Prebiotic Formula Delta; inoculating the growthbroth with the probiotic organism; growing the probiotic organism in thegrowth broth; harvesting the probiotic organism from the growth broth;selecting an agar spread plate for growing the probiotic organism,wherein the agar spread plate media is comprised of 1%-15% PrebioticFormula Delta; inoculating the agar spread plate with the probioticorganism harvested from the growth broth; growing the probiotic organismon the agar spread plate; and harvesting the probiotic organism from theagar spread plate.
 17. The method of claim 16, further comprising:freeze-drying the probiotic organism.
 18. The method of claim 16,further comprising: freeze-drying the probiotic organism by use of aFreeze-drying Buffer B when the probiotic organism is a probioticorganism selected from the group consisting of Bacteroides fragilis,Bacteroides ovatus, Bacteroides uniformis, Collinsella aerofaciens, andRoseburia hominis.
 19. The method of claim 16, further comprising:freeze-drying the probiotic organism by use of a Freeze-drying Buffer Cwhen the probiotic organism is Clostridium symbiosum.